Project Summary Our laboratory has developed novel culture methods using engineered Notch ligands to increase the number of CD34+ precursors, including those able to provide rapid hematopoietic engraftment in patients undergoing cord blood (CB) transplantation. Clinical trials indicate the need to derive greater hematopoietic stem and progenitor cell (HSPC) numbers for a more potent, economically feasible therapy. Consequently, this grant aims to elucidate new strategies to regulate Notch-induced signal strength to maximize HSPC growth, focusing on exploiting differences in 1) Notch receptor susceptibility to activation among HSPC subsets and 2) Notch signal strength necessary to promote HSPC expansion versus T cell differentiation. Towards this goal, we have determined that antibodies raised against the amino-terminus of the Notch extracellular domain are more effective than Notch ligands in activating receptors on primitive HSPC, leading to increased generation of in vivo repopulating cells; preliminary studies have suggested this results from antibody-mediated subversion of inhibitory effects imposed by endogenously expressed ligands in primitive HSPC (cis-inhibition). Thus, in Aim 1, we test our hypothesis that differential agonist potency in promoting Notch activation and HSPC generation results from ligand-mediated cis-inhibition. We will investigate inhibitory ligand expression and function in CB HSPC using shRNA knockdown and/or blocking antibody treatment together with transplantation assays. To elucidate the mechanism whereby antibody subverts cis-inhibition, guiding the future development of improved agonists, we will determine the contribution of target epitope location and increased agonist affinity. Our previous studies have also determined requirements for distinct levels of Notch signaling for generation of HSPC versus promotion of T cell differentiation. Thus, in Aim 2, we test our hypothesis that the use of paralog- specific Notch antibody agonists will fine-tune the level of Notch activation to concurrently enhance the generation of marrow and thymic repopulating cells. We will investigate the dynamic use of low densities of Notch paralog-specific antibody agonists to further increase marrow repopulating cell generation by inducing Notch activation in cis-inhibited primitive precursors, yet maintain a low level of Notch signal strength to prevent diversion of these cells to the T-lineage. We further explore whether paralog-specific antibody agonists can simultaneously generate marrow and thymic repopulating cells by inducing different levels of Notch activation in different hematopoietic subsets. Overall, these studies will provide insight into the cell- autonomous mechanisms regulating Notch receptor function, an outcome of conceptual and practical interest to those focused on manipulating cell-fate decisions in Notch-dependent stem cell types, most relevant here, the expansion of HSPC and prothymocyte precursors for the development of viable clinical therapeutics.